1. Field of Invention
The present invention relates to polyketide synthase and, more specifically, to a system and method for the heterologous expression of polyketide synthase gene clusters.
2. Background of Art
Natural products have been a major source of new drugs in all therapeutic areas. In some therapeutic areas, such as anti-bacterial and anti-cancer agents, greater than 70 percent of drugs approved between 1981 and 2002 have been derived from or inspired by natural products. In addition natural products serve as tools to better understand drug targets and pathways in human disease. By interacting with novel targets, natural products validate these targets for drug discovery, enabling diversification of the pharmacopoeia.
Bacterially derived polyketides and non-ribosomal peptides have played an extremely important role in natural product based drug discovery, particularly in the areas of antibiotic and anticancer agents. For example, one of the most recently approved anticancer agents, ixabepilone, is derived from the mixed polyketide non-ribosomal peptide epothilone B. In addition to antibiotic and anticancer activities, polyketides and non-ribosomal peptides have been shown to possess bioactivity in all major therapeutic areas.
Bacterial natural products, including polyketides and non-ribosomal peptides, offer key advantages in identifying biologically active small molecules. Because these compounds have evolved to offer their hosts increased fitness over other competing organisms, natural products have been pre-selected to target biological systems. As a direct result, natural products generally have appropriate solubility and stability in biologically relevant environments such as serum and cytoplasm and are often able to cross biologically relevant membranes. Natural products have been shown to offer unmatched chemical diversity and structural complexity as compared to de novo small synthetic molecules. It has been estimated that natural products libraries will have approximately a two orders of magnitude greater “hit rate” in identifying lead compounds than synthetic compound libraries.
Isolation of new bioactive polyketides for drug discovery and production of known polyketides for drug development is often limited, however, by the ability of the producing organisms to generate sufficient quantities of the desired compound. In addition, the traditional natural product screening approach used for the development of therapeutics has serious, inherent limitations, including building and maintaining high-quality natural product libraries, the frequent rediscovery of known compounds during the screening process, and difficulties associated with obtaining sufficient amounts of potential lead compounds for further evaluation. Despite the proven track record and intrinsic advantages of natural products in drug discovery, the majority of pharmaceutical companies downgraded or terminated their natural product research programs over the last twenty years. A driving force in this change was the difficulties associated with building and maintaining high quality natural product libraries. Isolation of natural products, which are normally trace components, from producing organisms is a challenging task and generally produces small quantities of compound. In addition the natural sources often cannot provide sufficient material for clinical develop of lead compound due to the limited sustainability of harvesting the producing organism. By the late 1980s, the natural products isolated were often known compounds leading to high rates of rediscovery. Rediscovery severely limited the accessible diversity of natural product libraries.
The inherent diversity of bacterially derived polyketide and non-ribosomal peptide natural products has not yet been tapped. Over one million bacterial strains have been cultured and screened for bioactive metabolites, leading to hundreds of clinically approved drugs and thousands of bioactive compounds. This represents less than 1 percent of the potential diversity available from bacterial sources. Genome sequencing has demonstrated that bacterial species generally posses gene clusters encoding for approximately twenty biosynthetic pathways. For culturable organisms traditional screening approaches have yielded two to three metabolites per species. Extrapolation from these data suggest that there are coding sequences for upwards of 200,000 natural products present in a single soil sample and that current screening approaches only access 200 to 500 compounds. Tapping this enormous natural product diversity will require a culture independent approach to compound screening.
Due to their important pharmaceutical role, methods for efficient production of polyketides are also highly desired. New methods for the discovery of cryptic or silent polyketides from biological pathways that are present yet not expressed have also increased interest in drug discovery. Complexities in the structure and stereochemistry of polyketides, however, limit synthetic production methods and have increased interest in microbial fermentation. Microbial fermentation as a means of production of polyketides has become a prominent economical method, but 99.8% of microbes available in native environments are not readily culturable or produce unacceptably low amounts of polyketides. The heterologous expression of secondary metabolites in more amenable heterologous hosts has recently become an attractive method for the production of polyketides. This approach has been used in the production of epothilone D for anticancer clinical trials. Heterologous expression of polyketide biosynthetic pathways proves difficult due to the complexity of the enzymatic pathways and the need for simultaneous expression of all genes in the pathway. These obstacles have prevented the expression of polyketides from poor fermentation hosts in the fermentation workhorse, Escherichia coli. 
Polyketides are biosynthesized by complex pathways containing multiple polyketide synthases, tailoring enzymes, and enzymes mediating resistance to the polyketide product. Oxytetracycline, as seen in FIG. 1B, which is produced by S. rimosus, provides an archetypical example of an aromatic polyketide synthase biosynthetic pathway. The backbone of oxytetracycline is assembled from a minimal PKS consisting of four proteins a ketosynthase (KS), a chain length factor (CLF), an acyl transferase (AT), and an acyl carrier protein (ACP), as seen in FIG. 1A. The minimal PKS catalyses repetitive Claisen-like condensations using ten sequential units of malonyl-CoA. Following backbone production, a minimum of six tailoring enzymes complete oxytetracycline biosynthesis. Overall the biosynthesis of oxytetracycline requires over a dozen enzymes working in concert.
The heterologous expression of biosynthetic pathways, such as the oxytetracycline biosynthetic pathway, require the heterologous host to recognize the promoters upstream of the biosynthetic genes and express the proteins. Both myxobacteria and streptomycetes have demonstrated an ability to recognize a broad range of promoters as displayed by their ability to produce an enormous number of polyketide products 4. Myxobacteria are the third largest producers of secondary metabolites behind actinomycetes and bacilli. Heterologous expression of a myxobacterial secondary metabolite has been observed in a streptomycetes host providing evidence for streptomycetes transcriptional machinery's ability to recognize and use myxobacterial promoter regions. However expression of a secondary metabolite from a streptomycetes has not been observed in a myxobacterium host.
Two approaches have been used to ensure expression of a biosynthetic pathway in a heterologous host, however both are limited in their scope and applicability. The first approach involves the selection of a host strain highly related to the native strain. By using highly related strains, the transcriptional machinery is expected to be conserved and therefore able to transcribe the heterologous genes. This approach has proven successful for the heterologous expression of polyketide gene clusters from the genus streptomyces. However, it is limited to producing polyacetate-derived polyketides. Additionally when gDNA from unrelated organisms are used the streptomyces-based heterolgous expression system fails to generate useful quantities of product. This approach is thus not expected to perform well for heterologous expression of pathways from diverse organisms, such as unculturable bacteria.
A second approach to ensuring heterologous expression is to replace the native promoters in a biosynthetic pathway with promoters known to function in the host strain. Both polyketide and non-ribosomal peptides have been expressed under the control of the T7 promoter in Escherichia coli. This approach is not well suited to screening large libraries of unsequenced gDNA as it is extremely labor intensive and requires foreknowledge of the genes in the biosynthetic pathway.